Borrelia channel-forming proteins : structure and function

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Borrelia channel-forming proteins:

structure and function

Ignas Bunikis

Department of Molecular Biology

Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå University

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Cover: Model of predicted BesC, BesA and BesB structures by Ignas Bunikis ISSN 0346-6612

ISBN 978-91-7264-971-2 Printed by Print & Media Umeå, Sweden 2010

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III | P a g e To my parents

Skiriu tėvams

“May the Force be with you.”

Yoda

"No problem! :)"

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Abstract

Borrelia is a Gram-negative, corkscrew-shaped bacterium transmitted by

infected ticks or lice. Borreliae are subdivided into pathogens of two diseases: Lyme disease, caused mainly by B. burgdorferi, B. afzelii and B. garinii; and relapsing fever caused primarily by B. duttonii, B. hermsii, B. recurrentis or B.

crocidurae. Both diseases differ in their manifestations, duration times and

dissemination patterns. Antibiotics are the major therapeutics, although unfortunately antibiotic treatment is not always beneficial. To date, drug resistance mechanisms in B. burgdorferi are unknown. Transporters of the resistance-nodulation-division (RND) family appear to be involved in drug resistance, especially in Gram-negative bacteria. They consist of three components: a cytoplasmic membrane export system, a membrane fusion protein (MFP), and an outer membrane factor (OMP). The major antibiotic efflux activity of this type in Escherichia coli is mediated by the tripartite multidrug resistance pump AcrAB-TolC. Based on the sequence homology we conclude that the besA (bb0140), besB (bb0141) and besC (bb0142) genes code for a similar efflux system in B. burgdorferi. We created a deletion mutant of

besC. The minimal inhibitory concentration (MIC) values of B. burgdorferi

carrying an inactive besC gene were 4- to 8-fold lower than in the wild type strain. Animal experiments showed that the besC mutant was unable to infect mice. Black lipid bilayer experiments were carried out to determine the biophysical properties of purified BesC. This study showed the importance of BesC protein for B. burgdorferi pathogenicity and resistance to antibiotics, although its importance in clinical isolates is not known.

Due to its small genome, Borrelia is metabolically and biosynthetically deficient, thereby making it highly dependent on nutrients provided by their hosts. The uptake of nutrients by Borrelia is not yet completely understood. We describe the purification and characterization of a 36-kDa protein that functions as a putative dicarboxylate-specific porin in the outer membrane of Borrelia. The protein was designated as DipA, for dicarboxylate-specific porin A. DipA was biophysically characterized using the black lipid bilayer assay. The permeation of KCl through the channel could be partly blocked by titrating the DipA-mediated membrane conductance with increasing concentrations of different organic dicarboxylic anions. The obtained results imply that DipA does not form a general diffusion pore, but a porin with a binding site specific for dicarboxylates which play important key roles in the deficient metabolic and biosynthetic pathways of Borrelia species.

The presence of porin P66 has been shown in both Lyme disease and relapsing fever spirochetes. In our study, purified P66 homologues from Lyme disease species B. burgdorferi, B. afzelii and B. garinii and relapsing fever species B.

duttonii, B. recurrentis and B. hermsii were compared and their biophysical

properties were further characterized in black lipid bilayer assay. Subsequently, the channel diameter of B. burgdorferi P66 was investigated in more detail. For this study, different nonelectrolytes with known hydrodynamic radii were used. This allowed us to determine the effective diameter of the P66 channel lumen. Furthermore, the blockage of the channel after addition of nonelectrolytes revealed seven subconducting states and indicated a heptameric structure of the P66 channel. These results may give more insight into the functional properties of this important porin.

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Papers in this thesis

This thesis is based on the following articles and manuscripts, which will be referred to by their Roman numerical (I-V).

I. An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds.

Bunikis I, Denker K, Östberg Y, Andersen C, Benz R, Bergström S.

PLoS Pathog. 2008 Feb 29;4(2):e1000009.

II. Oms38 is the first identified pore-forming protein in the outer membrane of relapsing fever spirochetes.

Thein M, Bunikis I, Denker K, Larsson C, Cutler S, Drancourt M, Schwan TG, Mentele R, Lottspeich F, Bergström S, Benz R.

J Bacteriol. 2008 Nov;190(21):7035-42. Epub 2008 Aug 29.

III. DipA, a pore-forming protein in the outer membrane of Lyme disease

Borrelia exhibits specificity for the permeation of dicarboxylates.

Thein M, Bonde M*, Bunikis I*, Denker K, Sickmann A, Bergström S, Benz R.

(Submitted)

IV. P66 porins are present in both Lyme disease and relapsing fever spirochetes: a comparison of the biophysical properties of P66 porins from six Borrelia species.

Barcena-Uribarri I, Thein M, Sacher A, Bunikis I, Bonde M, Bergström S, Benz R.

Biochim Biophys Acta. 2010 Feb 24.

V. The use of nonelectrolytes as molecular tools reveals the channel size and the oligomeric constitution of the Borrelia burgdorferi porin P66. Barcena-Uribarri I, Thein M, Maier E, Bonde M, Bunikis I, Bergström S, Benz R.

(Manuscript)

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Papers not included in this thesis

Contribution has been made by the author to the following articles that are not included in this thesis:

The etiological agent of Lyme disease, Borrelia burgdorferi, appears to contain only a few small RNA molecules.

Östberg Y, Bunikis I, Bergström S, Johansson J. J Bacteriol. 2004 Dec;186(24):8472-7.

Tickborne relapsing fever diagnosis obscured by malaria, Togo.

Nordstrand A, Bunikis I, Larsson C, Tsogbe K, Schwan TG, Nilsson M, Bergström S.

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Introduction

Borrelia is a fascinating organism! So is the research on this bacterium! It

reminds a little bit of jigsaw puzzle. The only big difference is that this particular puzzle does not have a defined size or a known number of pieces... Since its discovery, scientists have been captivated by this pathogen. In addition to many other studies, throughout the years hard and extensive work has been done in studying and understanding epidemiology, entomology, spirochete morphology, disease progression, its complex life cycle, virulence mechanisms, phenomenal antigenic variation, metabolism, host-pathogen interactions, genome organization, molecular mechanisms underlying cell processes, structural and biophysical properties of smallest building blocks of Borrelia. Each research group, each scientist contributes in solving fragments of a big picture by piecing together knowledge, ideas, experiments and discoveries made every day. In this thesis I present a few small puzzle pieces with the hope that they can fill in some gaps in the knowledge of Borrelia outer membrane components, their role in the pathogenicity and virulence of this spirochete. Perhaps one day, the combined efforts of many researchers around the globe will reveal the complete picture of this intriguing and unique bacterial species when all the pieces of the puzzle fall into place. But till then, there are many unsolved mysteries to be dealt with, many unanswered or unasked question to be answered or asked, and many discoveries to be made. There's plenty of research to be done, no question about that!

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Lyme disease and relapsing fever

History

The first known case of acrodermatitis chronica atrophicans, a late skin manifestation of Lyme borreliosis was described in 1883 by German physician Buchwald (Buchwald 1883). Later, a case of erythema migrans, the characteristic skin rash at the initial stage of Lyme borreliosis, was described by the Swedish dermatologist Arvid Afzelius, who suggested that the reason for the rash was the bite of a tick or another insect (Afzelius 1921). The first description of Lyme arthritis was reported in 1977 when Steere and coworkers described an unusually high incidence of acute arthritis among children in Old Lyme, Connecticut (Steere, Malawista et al. 1977). Several patients reported the development of a skin rash before the onset of arthritis. Because of the geographical clustering of the cases in the wooded areas and peak occurrence in the summer, Steere et al. postulated that the symptoms arose from transmission of an agent by an arthropod vector. A few years later, in 1981, while looking for rickettsiae, William Burgdorfer encountered long, irregularly coiled spirochetes when examining the midguts of Ixodes scapularis ticks (Burgdorfer 1984). In 1982, Burgdorfer and coworkers reported the correlation between the spirochete and Lyme disease (Burgdorfer, Barbour et al. 1982). In honor of its discoverer, the Lyme disease spirochete was named Borrelia

burgdorferi (Johnson, Schmid et al. 1984). To acknowledge early findings of the

pioneer Afzelius, a common etiological agent of Lyme disease in Europe was named B. afzelii (Canica, Nato et al. 1993).

In contrast to Lyme disease, relapsing fever has been recorded and described since ancient times, even though the disease had not been given its contemporary name and the causative agent had not yet been determined. An epidemic fever was described by Hippocrates, called the "ardent fever", and during the following centuries preceding modern times, several outbreaks of epidemic fever had been suggested to be relapsing fever (Felsenfeld 1971). In middle of the 18th century, the first clinical features were well described in a documented outbreak in Ireland. This epidemic spread over the British Islands, and the disease was for the first time designated "relapsing fever" in 1843 (Southern and Sanford 1969; Felsenfeld 1971). From England, relapsing fever was introduced to the European continent and the United States, causing

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3 | P a g e outbreaks in populations living under poor conditions. The organism causing the epidemic disease was discovered in 1868 by the German physician Obermeier, and was named Spirocheta obermeieri, but later on the organism was designated Borrelia recurrentis (Felsenfeld 1971). The biggest outbreaks of epidemic relapsing fever occurred during World War I and II when hundreds of thousands soldiers and civilians were infected. A similar disease was described on the African continent for the first time by the famous explorer Dr. Livingstone in 1857, but it was not until the beginning of the 20th century that presence of Borrelia in blood samples was noticed. Relapsing fever has since then been reported in almost all parts of the world, with some exceptions (Felsenfeld 1971).

Clinical manifestations

Clinically, Lyme disease is most similar to syphilis, both exhibiting multisystem involvement and mimicry of other diseases (Strle, Picken et al. 1997). The most common sign of Lyme borreliosis is a red skin rash called erythema migrans observed in 80-89% of all the clinical cases in Europe and North America (Steere 1989; Mehnert and Krause 2005). The erythema migrans expands from the site of tick bite with central clearing, and without treatment it can reach one meter in diameter (Stanek and Strle 2003). The skin lesion is often accompanied by influenza-like symptoms such as nausea, fatigue, fever and headache. This is followed by a disseminated infection within days or weeks affecting the nervous system, joints, skin and heart. Subsequently, a persistent infection can evolve within weeks or months (Steere 2001). European patients can develop a chronic skin manifestation called acrodermatitis chronica atrophicans. Live spirochetes have been isolated from such lesions more than 10 years after the onset of the disease (Asbrink and Hovmark 1985). In about 10% of the patients, no symptoms of infection can be seen (Steere 2001).

In contrast to Lyme borreliosis, relapsing fever is an acute disease with a high degree of bacteremia. The incubation period is 2-18 days after the tick bite. An infected patient develops a sudden high fever, accompanied by chills, severe headache and other undefined flu-like symptoms. Borrelia spirochetes are highly invasive and can be found in variety of tissues, although spleen and liver are affected the most during early infection. The symptomatic period of

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relapsing fever lasts for 3-5 days. Thereafter, the patient experiences a pronounced temperature elevation, followed by a sudden temperature drop down to normal or even lower level than pre-infection. The febrile period is a result of massive bacteremia as the Borrelia multiplies in the blood, reaching as many as 108_{bacteria per milliliter of blood. After the body temperature has}

gone down to normal, a period of well-being occurs when no bacteria are detected in the blood. An afebrile period lasts for 4-7 days whereafter the disease relapses with subsequent fever peaks and spirochetemia. The first attack is commonly more severe and prolonged than the following relapses (Southern and Sanford 1969). Borrelia can penetrate the blood-brain barrier and infect brain, which can be manifested by meningitis, facial palsy and neuritis (Cadavid and Barbour 1998). In addition, relapsing fever is associated with a high incidence of pregnancy complications, such as spontaneous abortions and preterm delivery (Jongen, van Roosmalen et al. 1997).

Detection and treatment

Tick bite history and general awareness after staying in a Lyme borreliosis endemic area are crucial for diagnosis. Persons who developed a skin lesion or other illness within one month after removing an attached tick should consult a physician. The erythema migrans is still one of the most easily noticeable signs of an infection (Wormser, Nadelman et al. 2000). Detection of Borrelia by polymerase chain reaction (PCR) is useful, but the method has a major drawback since it cannot distinguish between living and dead bacteria. However, it can be the method of choice in later stages of disease (Steere 2001). Isolation of bacteria in culture is the ultimate proof, although it is time-consuming, has low sensitivity and depends on infectious species and the stage of disease (Nadelman and Wormser 1998; Wilske 2005). Antibody detection of surface localized antigens is a common, constantly improving method, but cannot be applied at the initial stages of the disease (Steere 2001). When used, it is recommended that positive Enzyme-Linked Immunosorbent Assay (ELISA) results are confirmed by Western blot analysis (Wilske 2005).

Due to diffuse first symptoms resembling influenza, relapsing fever can be difficult to diagnose. Recently, it has been shown that relapsing fever can be obscured by malaria infection (Nordstrand, Bunikis et al. 2007). Prevalence of

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5 | P a g e malaria in the same areas as relapsing fever and symptomatic similarities of both diseases can lead to misdiagnosis and mistreatment. In addition, very often a tick bite is left unnoticed because of the nocturnal lifestyle of the soft-bodied ticks transmitting Borrelia and their ability to complete the blood-meal in as fast as a few minutes. Together with the fact that relapsing fever does not cause erythema migrans, the start of the disease becomes difficult to detect.

The easiest way to detect ongoing relapsing fever infection is by examining a blood sample from the infected patient, since the Borrelia could be detected immediately by dark-field microscopy or by Wright-Giemsa staining. However, such detection requires a sufficient number of bacteria in the blood, such as during the first peak of spirochetemia, a time period that is very easily missed. The sensibility of this method could be improved by removing the red blood cells and concentrating the spirochetes in the plasma (Larsson and Bergström 2008). Serological tests are available, although such tests have limited sensitivity due to high antigenic variation and cross-reactivity with other Borrelia species. GlpQ, a protein that is conserved among all members of relapsing fever Borrelia, but is absent in Lyme disease spirochetes, can be used to discriminate between these two agents (Schwan, Schrumpf et al. 1996; Nordstrand, Bunikis et al. 2007). Even though serological diagnosis of relapsing fever is possible, the time point for effective treatment could be missed. The antibody response towards the bacterium develops after approximately 4-6 days (Yokota, Morshed et al. 1997), sufficient time for Borrelia to disseminate to tissues such as brain with limited access for antibiotics (Andersson, Nordstrand et al. 2007).

In most cases, use of antibiotics against Borrelia results in successful treatment (Bryceson, Parry et al. 1970). Commonly used antibiotics are penicillin, erythromycin and tetracycline. The clinical manifestations and age of the patient can influence type and length of the antibiotic treatment (Wormser, Nadelman et al. 2000). In the case of relapsing fever, it should be kept in mind that treatment with antibiotics can cause a life-threatening Jarish-Herxheimer reaction provoked by release of bacterial components when bacteria are lysed by antibiotics (Bryceson 1976).

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Prevention

The most effective approach to prevent and reduce the incidence of Lyme disease is to avoid contact with infected ticks. Limited outdoor activities in tick infested areas and properly tucked clothes not leaving any exposed skin provide effective protection. The risk of attracting ticks also can be reduced by application of mosquito or tick repellents. Still, removing any attached ticks within 24 hours of attachment reduces the chance of infection dramatically. It is therefore important to examine the body for ticks when staying in endemic areas (Piesman, Mather et al. 1987). Although difficult, reducing the vector population through habitat manipulation and insecticides is possible. Another approach is vaccination. Unfortunately, currently there is no vaccine available against Lyme borreliosis. However, a vaccine called LYMErix was commercially available in the USA from 1998 but withdrawn from the market in 2002 due to rare cases of vaccinated persons developing autoimmune arthritis, although this was never proven (Steere 2006). The vaccine was based on recombinant outer surface protein A (rOspA) and conferred protection in a unique way. Bactericidal antibodies generated against OspA eliminate B. burgdorferi within the tick during feeding, preventing infectious spirochetes from entering the host (Fikrig, Telford et al. 1992). LYMErix was only effective against B. burgdorferi infections, and therefore availability was restricted to the USA, where this genospecies alone is responsible for all clinical cases. In Europe and Asia, B. burgdorferi is less common. Instead, B. garinii and B. afzelii are the major causative agents for Lyme borreliosis. A vaccine for the European market therefore needs to provide protection against all three genospecies. Now LYMErix vaccine is available for dogs only (Ma, Hine et al. 1996). Different attempts to identify new, suitable vaccine candidates have been tried through the years, but this has not yet led to any commercially available vaccine.

The situation is similar for relapsing fever, currently there is no vaccine available. Therefore human infection can only be avoided by reducing or eliminating the vectors that transmit the disease. This can be difficult in areas where the vector lives in close association with humans. Relapsing fever transmitting ticks live in the cracks and crevices in over 80% of mud-huts (Barclay and Coulter 1990), a common type of house in sub-Saharan Africa (Sonenshine 1997). Different repellants and bed-nets have been used and proven to be successful in reducing exposure of humans to the ticks

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7 | P a g e (Sonenshine 1997). Fighting against louse-borne relapsing fever is even more difficult. Improvement of living conditions and hygienic standards would help eradicate the louse, although in areas affected by poverty this is a very hard task.

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Borrelia

Classification

Borrelia belongs to the Spirochete phylum in the class of Spirochaetes and the

order of Spirochetales. Examples of other human disease-causing spirochetes are Treponema pallidum (causing syphilis), T. denticola (periodontal diseases) and Leptospira interorrgans (leptospirosis) (Paster and Dewhirst 2000). The genus Borrelia is comprised of many different genospecies, but the two groups causing disease in humans are Lyme Borrelia genospecies and relapsing fever

Borrelia genospecies.

The Lyme disease Borrelia group of spirochetes includes 12 known genospecies.

B. burgdorferi, B. garinii and B. afzelii are known to cause disease in humans

(Stanek and Strle 2003). Lately, a few clinical cases due to B. bissetii, B.

lusitaniae and B. spielmanii infections also have been reported (Maraspin,

Cimperman et al. 2002; Collares-Pereira, Couceiro et al. 2004; Fingerle, Schulte-Spechtel et al. 2008). The tick vector, geographical distribution and reservoir animals differ between the genospecies. In North America, clinical cases of Lyme borreliosis are due to infection of B. burgdorferi. In Europe and Asia the situation is more complex due to three causative agents, namely B. garinii, B.

afzelii and B. burgdorferi, although the latter is not very common in Asia

(Kurtenbach, Hanincova et al. 2006). In addition, these genospecies are spread by two different vectors: Ixodes ricinus in Europe and I. persulcatus in Asia. The habitats of these two ticks overlap in Eastern Europe (Kurtenbach, Hanincova et al. 2006). Lyme disease Borrelia can be genotyped using a phenotypic method which includes monoclonal antibodies reacting against different classes of the outer surface protein A (OspA). Using this method, seven OspA serotypes have been identified (Wilske, Preac-Mursic et al. 1993). OspC has also been the basis of classification with monoclonal antibodies, separating Lyme Borrelia into additional thirteen classes (Wilske, Jauris-Heipke et al. 1995). In recent years, the phenotypic typing methods are being replaced in favor of genotypic methods based on DNA sequence comparison. Genes used for genotyping are

p66 (porin), flaB (a structural protein of flagella), ospA and ospC (Assous, Postic

et al. 1994; Pinne, Thein et al. 2007). The drawback using these genes is the high level of sequence similarity among different genospecies. In a comparative study the intergenic spacer (IGS) located on the chromosome between rrs (16S)

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9 | P a g e and rrlA (23S) genes was identified as the most suitable locus for genospecies identification and evolutionary studies since this intergenic sequence is not under selective pressure and therefore is highly polymorphic (Bunikis, Garpmo et al. 2004).

The relapsing fever group is further divided into "new world" and "old world"

Borrelia species based on the continent in which their vectors exist. Old world

species are present on the European, African and Asian continents, whereas new world species are defined as those present in South and North America. Many known relapsing fever species are classified according to their association with their arthropod vector. The ticks are adapted to live in their specific microhabitat, thereby making the Borrelia also specific for a certain geographic location (Sonenshine 1997). The tick-Borrelia relationship is in many cases so specific that one Borrelia species cannot be transmitted by the vector specific for other Borrelia species (Barbour and Hayes 1986). There has also been a division of species based on the range of mammals that they infect. For example, louse-borne B. recurrentis is considered to be a strictly human-specific pathogen. New technologies and genotyping based on DNA sequencing lead to a revision of the phylogeny. The 16S rRNA gene has been used to compare and distinguish different species, although due to a high degree of conservation and only minor sequence differences, it is hard to separate closely-related organisms (Fox, Wisotzkey et al. 1992; Ras, Lascola et al. 1996). Therefore, the above described genotyping based on intergenic spacer sequence is a valuable alternative.

General characteristics

The Borrelia spirochetes have an obligate parasitic lifestyle whereby they are only able to survive in a tick vector or a vertebrate host. They are coiled, up to 30 µm long and less than 1 µm thick (Figure+1) (Goldstein, Buttle et al. 1996). Variation can, however, occur between genospecies. The generation time is determined to be on average 5 to 12 hours in vitro during the logarithmic growth phase at the optimal temperature of about 35°C, which is extremely long compared to other bacteria (Barbour 1984). The bacterium consists of an outer membrane with numerous lipoproteins on the surface, a peptidoglycan layer in the periplasmic space, and an inner membrane that surrounds the

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cytoplasm (Barbour and Hayes 1986). All Borrelia species are highly motile and move by way of a spinning/wave-like motion by rotating their flagella clockwise or counter-clockwise (Goldstein, Charon et al. 1994). The flagella are inserted at the ends of the bacterium and, like the cytoskeleton of an eukaryotic cell, they render the spirochetes wave-like in appearance (Wolgemuth, Charon et al. 2006). The number of flagella differs between different species and can be as many as 30 (Charon, Greenberg et al. 1992). The lack of flagella results in a long rod-shaped non-motile bacterium (Motaleb, Corum et al. 2000). Although

Borrelia have both inner and outer membranes and are generally considered to

be Gram-negative bacteria, they have many features similar to Gram-positive bacteria such as the absence of lipopolysaccharide (LPS) and presence of membrane glycolipids (Takayama, Rothenberg et al. 1987; Hossain, Wellensiek et al. 2001). Borrelia has a very segmented genome with a linear chromosome and high number of circular and linear plasmids, which carry genes that are necessary for the bacteria to fulfil the infection cycle. The total size of the genome does not exceed 2M base pairs and encodes limited biosynthetic genes, suggesting that many nutritional components are taken up from the host (Fraser, Casjens et al. 1997; Casjens 2000). This makes the bacterium difficult to culture and a complex medium, Barbour-Stoenner-Kelly II (BSKII) (Barbour 1984), supplemented with rabbit serum is needed.

Figure 1. Borrelia burgdorferi. Photo taken by Ignas Bunikis in the year 2004, half a year before starting his PhD studies.

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Transmission and adaptation

Lyme disease and relapsing fever are zoonotic diseases, transmitted by the arthropod vector to different vertebrate hosts (Barbour and Hayes 1986). The bacteria are strictly dependent on the arthropod and the reservoir host to be maintained in nature, and have therefore a biphasic life cycle where they alternate between infecting the vector and the vertebrate. The success of this alternating lifestyle is dependent on adaptation to different environments. Several features contribute to the success of ticks as vectors for pathogen transmission: a long lifespan and durability, as they can resist starving for years, and an ability to infect a majority of vertebrates (Sonenshine 1997). During its life cycle, a tick goes through three developmental stages: the larva, the nymph and the adult stage. All three developmental forms require blood-meals before transformation to the next stage (Sonenshine 1997). The transmission of

Borrelia occurs when a naϊve larva, nymph or tick feeds on a Borrelia-infected

animal. Borrelia replicates in the midgut and when their number increases, they migrate towards the tick internal organs, such as nervous tissue, the salivary glands and the coxal organs (Barbour and Hayes 1986). The bacteria can persist in the tick vector for several years before they are transmitted to a vertebrate host with the blood-meal (Sonenshine 1997).

Ticks transmitting Borrelia are divided into two major families: the hard-bodied tick (Ixodidae) and the soft-bodied tick (Argasidae). The hard-bodied ticks are vectors for Borrelia species causing Lyme disease. Ixodes tick feeding is a slow process and can last from 3–5 days for a larva, up to 4–7 days for a nymph and 7–11 days for an adult tick (Sonenshine 1997). To be able to infect a new host, the spirochetes need to penetrate the epithelium of the tick midgut and migrate through the hemolymph to the salivary glands. In the case of the nymph, the migration process takes 36-48 hours and starts when the tick begins to feed and blood fills up the midgut (Ribeiro, Mather et al. 1987; De Silva and Fikrig 1995). During the first 24 hours of nymphal attachment, virtually no transmission of spirochetes to the host takes place. After 48 hours transmission, occurs but particularly efficient transmission takes place only after 72 hours (Piesman, Mather et al. 1987). Removing a feeding tick within 24 hours after attachment is therefore an efficient way of preventing spread of spirochetes to a new host. Tick feeding triggers a shift in expression of major outer surface proteins of

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lipoprotein anchored to the bacterial outer membrane (Schwan, Piesman et al. 1995). OspA has been shown to mediate adherence to the tick midgut epithelium (Pal, Li et al. 2004). This interaction is essential for colonization of the tick gut. When the tick starts to feed on a host, the incoming blood changes the environment of the midgut. The OspA expression is ceased and, instead, up-regulated expression of another lipoprotein OspC occurs (Schwan, Piesman et al. 1995). The reciprocal expression of OspA and OspC correlates with exit of spirochetes from midgut, migration through the hemolymph to the salivary glands and entry into the new host. OspC is essential for the initial infection of the mammalian host (Grimm, Tilly et al. 2004).

The largest group of Argasidae family is the Ornithodoros, which is the main vector for relapsing fever. As all ticks, they need blood-meals for their development. Soft-bodied ticks live in close proximity to their vertebrate hosts and are the most active during the night. Ornithodoros ticks are fast feeders — they do not consume large volumes of blood at once, instead they can feed several times and on multiple hosts. This increases the spreading frequency of bacteria (Sonenshine 1997). When the infected tick feeds on a naϊve animal, the bacteria are deposited into the vertebrate skin, rapidly migrate into circulation and multiply there, reaching high numbers. The bacteria subsequently leave the circulation and penetrate various tissues such as the liver, spleen, kidneys or brain. B. recurrentis, causing epidemic relapsing fever, is transmitted by the human body louse Pediculus humanus humanus. Lice are relatively short-lived and molt three times before reaching adult stage. They typically feed five times per day (Raoult and Roux 1999). Louse-ingested Borrelia takes the route from the midgut to the hemolymph where it multiplies. Later it disseminates to all cavities of the louse body without entering the tissues (Southern and Sanford 1969). The transmission of B. recurrentis to humans does not occur through the bite, instead the bacteria are transferred when the louse is crushed and the spirochete-containing hemolymph is rubbed into the bite site (Barbour and Hayes 1986).

Early researchers noticed that every relapse of the disease was caused by a new variant of the original infecting strain. Eventually, it was evident that the relapsing feature was a result of antigenic variation of the dominant surface protein, the Vmp's (Barbour, Tessier et al. 1982; Stoenner, Dodd et al. 1982). During a fever peak, the majority of the spirochetes express one type of Vmp,

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13 | P a g e eliciting the host to mount an antibody response towards this serotype, subsequently clearing the organisms from the circulation. However, a second wave of bacteria, expressing a different Vmp, will multiply and cause the disease to relapse. Antigenic variation contributes to maintenance of Borrelia in nature since it prolongs bacterial survival in the circulation, thereby increasing the chance of successful transmission to a naϊve host (Barbour and Hayes 1986). In addition, Vmp's are involved in adaptation to various surroundings and determinants of tissue tropism (Cadavid, Thomas et al. 1994; Cadavid, Pachner et al. 2001).

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Genomes of

Borrelia

Borrelia genomes

Borrelia is genomically unique and not closely related to any other bacteria,

including the other spirochetes. B. burgdorferi and B. hermsii represent the two major branches within the Borrelia genus, wherein B. burgdorferi typifies the ‘Lyme disease agent-like’ branch and B. hermsii typifies the ‘relapsing fever agent-like’ branch (Paster, Dewhirst et al. 1991). Each Borrelia species carries a linear chromosome about 900 kbp in length and multiple circular and linear low copy number plasmids that are usually, but not always, in the 9–62 kbp range. The chromosomes of these bacteria all have quite similar gene contents, whereas the plasmids are more variable. Individual Borrelia cells harbor a more diverse plasmid complement than that of any other bacterium — the sequenced genome of B. burgdorferi type strain B31 has 21 plasmids, and several additional plasmids appear to have been lost between isolation of the strain in 1982 and the completion of its genome sequence (Casjens, Palmer et al. 2000). Overall, the 900-kbp chromosomes, often called the ‘large’ or ‘main’ chromosomes, carry the great majority of the genes that encode metabolic enzymes, and the 400 to 650 kbp of plasmid DNAs carry the bulk of the surface lipoprotein encoding genes. The chromosome carries tightly packed genes, as is typical of bacteria, while many of the linear plasmids have substantially lower gene densities and many apparently decaying pseudogenes (Casjens 2000; Casjens, Palmer et al. 2000).

The complete 910,725-bp sequence of the B. burgdorferi strain B31 linear chromosome was published in 1997 by Fraser et al. (Fraser, Casjens et al. 1997), and it remains the only Borrelia strain whose genome sequence is known to be complete (some plasmid sequences remain unassembled in the others). Its nucleotide composition is 28.6% G+C and is predicted to contain 846 apparently intact protein encoding genes and ten or fewer genes that appear to be truncated or contain frame shift mutations; the fraction of mutational inactivated chromosomal genes is among the lowest of the analyzed bacterial genomes. Although smaller bacterial genomes are known, this chromosome size is near the small end of the spectrum (Casjens 1998). The protein-encoding genes occupy 93% of the chromosome, a typical value for a bacterial genome not undergoing current reduction in size (Lynch 2006). These protein-encoding

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15 | P a g e genes are quite highly clustered according to function, often suggesting transcription as operons; about 67% of the genes are oriented such that they are transcribed away from the center of the chromosome. About 60% of the strain B31 chromosome’s predicted genes have some similarity to a gene in another organism whose role or function is at least partly understood; about 10% are similar to known genes in other organisms whose roles are unknown; and about 30% are unique to Borrelia and have unknown functions (Fraser, Casjens et al. 1997). The chromosome carries what appears to be a rather minimal set of the genes that are required for cell maintenance and replication. The biosynthetic and intermediary metabolic capacity of Borrelia is very limited. Genes that encode enzymes that perform functions in respiration, amino acid synthesis, nucleotide synthesis, lipid synthesis and enzyme cofactor synthesis are almost completely lacking, in agreement with their many and fastidious requirements for growth in culture and their evolutionary adaptation to two highly specific host environments. Given the requirement that they scavenge essentially all amino acids, nucleosides, lipids and cofactors from their environment, it is perhaps surprising that ‘only’ 16 transporters were identified in the initial analysis, and only a few more have been identified since then. Some of these transporters probably have generic specificity and have been hypothesized to bring in multiple related small molecules (Fraser, Casjens et al. 1997).

Plasmids and virulence

With the notable exception of cp26, the plasmids are not required for growth in culture (Sadziene, Wilske et al. 1993). A relationship between the plasmid content of B. burgdorferi and virulence has been well established. In B.

burgdorferi strain B31, linear plasmids lp25 and lp28-1 are necessary for high

infectivity in mice (Labandeira-Rey and Skare 2001; Purser, Lawrenz et al. 2003), while lp25 and lp28-4 are important for maintenance in ticks (Grimm, Tilly et al. 2005; Strother and de Silva 2005). Circular plasmid cp26 is found in all isolates and encodes OspC, which is required for establishment of infection in mice (Pal, Yang et al. 2004). Recently, lack of lp36 was shown to correlate with reduced infectivity in mice, and the defect could be partially complemented by the lp36-encoded adenine deaminase (Jewett, Byram et al. 2007). However, B.

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16 | P a g e

virulence in mice (Wang, Ojaimi et al. 2002), suggesting that additional virulence factors remain to be identified.

Genomic relationships

Since the publication of the B. burgdorferi strain B31 genome sequence, the complete nucleotide sequences of the chromosomes of B. garinii strain PBi and

B. afzelii strain PKo have been published along with the complete sequences of

some of their circular and linear plasmids (Glockner, Lehmann et al. 2004; Glockner, Schulte-Spechtel et al. 2006). In 2008, Lescot et al. reported the sequences of B. duttonii Ly and B. recurrentis A1 and some of their plasmids (Lescot, Audic et al. 2008). In addition, chromosome sequences for relapsing fever isolates B. hermsii DAH and B. turicatae 91E135 are available through GenBank. Data suggest that chromosomes of all the Borrelia spp. have highly similar gene orders. The analysis of the sequence of the B. garinii PBi and B.

afzelii PKo chromosomes showed that the two chromosomes are essentially

completely co-linear with the B. burgdorferi chromosome except for the regions very near the telomeres, and that the common regions of PBi and PKo are about 93% identical in nucleotide sequence (Glockner, Lehmann et al. 2004; Glockner, Schulte-Spechtel et al. 2006). PKo and PBi are about 93% identical whereas those of B31 and PKo are 91% identical, indicating that the three species, B.

burgdorferi, B. garinii and B. afzelii, are approximately equidistantly related.

Analysis of B. hermsii strain DAH and B. turicatae strain 91E135 chromosomal draft sequences showed that these two strains contain three purine metabolism genes that B. burgdorferi lacks (Pettersson, Schrumpf et al. 2007). In addition, the relapsing fever Borrelia chromosomes have two genes that encode enzymes for glycerol-3-phosphate acquisition that are not present in the Lyme disease spirochetes (Schwan, Battisti et al. 2003). Lescot et al. found only 13 genes unique to the Lyme agents and seventeen unique to the relapsing fever agents when their B. duttonii and B. recurrentis chromosome sequences were compared to the B. burgdorferi B31 chromosome sequence (Lescot, Audic et al. 2008). Thus, the relapsing fever and Lyme disease agent chromosomes are quite similar overall. The B. garinii PBi and B. afzelii PKo genomes carry eight and six linear plasmids, respectively, as well as at least three and nine circular plasmids, respectively. The circular plasmids are largely homologous to their B.

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17 | P a g e Glockner, Schulte-Spechtel et al. 2006). Among the linear plasmids, both PBi and PKo also carry plasmids that are very similar to B. burgdorferi lp54. The relapsing fever Borrelia also harbor numerous linear and circular plasmids in the same size ranges as those of the Lyme agents, and at least some isolates have one or two much larger linear plasmids in the 100–220 kbp size range (Lescot, Audic et al. 2008). The 180-kbp linear plasmid of B. hermsii HS1 carries nucleotide metabolism genes that are not present in the Lyme agent Borrelia genomes (Zhong, Skouloubris et al. 2006). Lescot et al. reported the sequences of seventeen B. duttonii and eight B. recurrentis plasmid sequence contigs (Lescot, Audic et al. 2008). From this work, it is clear that although the genes are similar, the gene organization of plasmids in relapsing fever Borrelia is often quite different from that in B. burgdorferi.

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Metabolism of

Borrelia

Limited de novo biosynthetic capacity of Borrelia

Borrelia burgdorferi is an obligate bacterial pathogen with no known ability to

survive outside of either the arthropod vector or mammalian host. This apparent inability to survive outside of a host implies that B. burgdorferi is restricted in terms of its metabolic capabilities and is consistent with its fastidious nature. On the other hand, the ability to survive in such disparate hosts as ticks and warm-blooded mammals implies that B. burgdorferi is adept at incorporating metabolites found in one or both of the arthropod and mammalian species they infect. Identification of first specific Borrelia porin involved putative nutrient uptake is described in Paper II and Paper III of this thesis. The genome sequence of B. burgdorferi indicates that the primary source for energy production is by fermentation via the Embden-Meyerhof pathway (Fraser, Casjens et al. 1997; Das, Hegyi et al. 2000; von Lackum and Stevenson 2005). No homologues of TCA enzymes or proteins involved in electron transport and oxidative phosphorylation are encoded in the genome (Fraser, Casjens et al. 1997; Das, Hegyi et al. 2000; von Lackum and Stevenson 2005). Despite the limited battery of enzymes involved in energy production, B.

burgdorferi is able to survive and persist for long periods of time inside the host

they colonize.

Carbon utilization

B. burgdorferi uses phosphotransferase system (PTS), a family of well

characterized sugar transporters, for acquisition of carbohydrates across the cytoplasmic membrane. It is predicted that Borrelia possesses six distinct PTS transporters which mediate the accumulation of glucose, N-acetylglucosamine, chitobiose, and mannose (Fraser, Casjens et al. 1997; Saier 2000; von Lackum and Stevenson 2005). Three of the six PTS transporters are thought to be competent for glucose uptake, suggesting that glucose is the preferred carbon source of B. burgdorferi. In addition to the PTS transporters, it is predicted that

B. burgdorferi utilizes an Mgl ABC system for glucose transport. Mannose and

chitobiose are predicted to be transported via PTS as well and used as alternative carbon sources (Fraser, Casjens et al. 1997; von Lackum and

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19 | P a g e Stevenson 2005). Although the genome sequence suggested that additional carbon sources, including fructose, galactose, and ribose, could be utilized, studies have shown Borrelia growth limitation in modified BSK media using these carbohydrates. It was determined that B. burgdorferi growth is restricted to glucose, maltose, N-acetylglucosamine, chitobiose, mannose, and glycerol as carbon sources during in vitro growth (von Lackum and Stevenson 2005). Most likely, Borrelia uses glucose to produce galactose, which is required for the synthesis of the major membrane glycolipid, α-monogalactosyl diacylglycerol (α-MgalDAG) (Östberg, Berg et al. 2007). This glycolipid has been proposed to be required for membrane stability and function (Mannock, Lewis et al. 2001). The homologues of all of the enzymes required for the oxidative phase of the pentose phosphate pathway are present within B. burgdorferi, while several enzymes are missing from the non-oxidative phase of the pentose phosphate pathway suggesting that ribose is probably required for RNA and riboflavin biosynthesis and not for generating energy. This would explain the presence in

Borrelia of a putative ribose transport system and inability to use it as a carbon

source during in vitro growth (Fraser, Casjens et al. 1997; von Lackum and Stevenson 2005).

The transport of chitobiose is of particular interest as it is a derivative of the chitin exoskeleton of the arthropod vector, specifically a β-1,4-linked disaccharide of N-acetyl glucosamine (NAG) that is not found in mammals. It was experimentally shown that B. burgdorferi can transport chitobiose, although the genetic inactivation of the chbC gene (chitobiose PTS transporter) did not affect growth in either the tick vector or mammalian hosts, indicating that other sugars present within the tick can compensate for the loss of this transport system (Tilly, Elias et al. 2001; Tilly, Grimm et al. 2004). Alternatively, a putative beta-glucosidase, encoded by bb0620, could function to cleave chitobiose to generate NAG molecules that can then be transported through PTS transporters (Fraser, Casjens et al. 1997). After NAG is phosphorylated, it can then either be incorporated into cell wall biosynthesis or deacetylated to generate glucosamine-6-phosphate that is then converted to fructose-6-phosphate, a critical intermediate substrate in the glycolytic pathway. In addition, it has been observed that NAG can serve as an alternative carbon source and support in vitro growth of B. burgdorferi (von Lackum and Stevenson 2005).

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The B. burgdorferi genome is predicted to contain an operon (bb0240 through

bb0243) encoding a glycerol transporter, enzymes that convert glycerol to

glycerol-3-phosphate and isomerize glycerol-3-phosphate to dihydroxyacetonephosphate. Once dihydroxyacetone phosphate is formed, it can isomerize into glyceraldehyde-3-phosphate, which then proceeds to the glycolytic pathway to generate ATP. In addition to its entry into glycolysis, glycerol-3-phosphate can be shuttled into phospholipid, lipoprotein and glycolipid biosynthesis (Fraser, Casjens et al. 1997; Saier 2000; von Lackum and Stevenson 2005).

ATP synthesis

B. burgdorferi lacks genes homologous to those encoding proteins involved in

oxidative phosphorylation or the TCA cycle (Fraser, Casjens et al. 1997; Das, Hegyi et al. 2000). They also do not appear to make energy via the pentose phosphate pathway, but instead use it to generate building blocks for DNA and RNA (von Lackum and Stevenson 2005). However, they do have all of the genes required for the glycolytic pathway. The major metabolic pathway for ATP generation in B. burgdorferi appears to be substrate-level phosphorylation mediated by the phosphoglycerate kinase and pyruvate kinase enzymatic steps during fermentation (Fraser, Casjens et al. 1997).

Most bacteria generate a proton gradient across their cytoplasmic membrane due to electron transport systems that result in a greater concentration of protons outside the cytoplasm. The protons then cross the concentration gradient through an F-type ATPase in a process that generates ATP within the cytoplasm of the bacterium. However, B. burgdorferi does not encode an F-type ATPase. In contrast to the majority of bacteria, it possesses a V-type ATPase which functions in reverse compared to F-type ATPases, and thus utilizes energy instead of synthesizing ATP (Fraser, Casjens et al. 1997). Most likely, the V-type ATPase in B. burgdorferi functions primarily as a H+ pump that establishes a proton motive force across the inner membrane, thereby driving some proton-coupled transport systems and B. burgdorferi motility. The exact function of this important protein complex remains to be experimentally determined.

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Cell envelope of

Borrelia

Borrelia membranes

The borrelial outer cell membrane is fluid and consists of 45–62% protein, 23– 50% lipid and 3–4% carbohydrate (Barbour, Hayes et al. 1986). Its composition differs significantly from that of Gram-negative bacteria. A first, particularly distinguishing, feature is the absence of phosphatidylethanolamine (Belisle, Brandt et al. 1994) and lipopolysaccharide (LPS) (Takayama, Rothenberg et al. 1987) and the presence of non-LPS glycolipid antigens (Eiffert, Lotter et al. 1991; Wheeler, Garcia Monco et al. 1993; Belisle, Brandt et al. 1994). These glycolipids represent about 50% of the total lipids and contain only galactose as the monosaccharide constituent (Hossain, Wellensiek et al. 2001). Secondly, the B.

burgdorferi outer membrane exhibits a relatively low density of

transmembrane-spanning proteins as determined by freeze fracture EM studies (Walker, Borenstein et al. 1991; Radolf 1994; Jones, Bourell et al. 1995). This may explain why Borrelia is more susceptible than Gram-negative bacteria to detergents or to disruption by routine physical manipulations such as centrifugation and resuspension. Thirdly, the outer membrane contains an unusually large number of lipoproteins (Brandt, Riley et al. 1990), many of which are on the bacterial surface, the host–pathogen interface, where they act as adhesins, targets for bactericidal antibodies, or receptors for various molecules. The phospholipid content of borrelial membranes differs from those of other bacteria, including other spirochetes, such as Treponema pallidum. Mass spectroscopic analysis identified α-monogalactosyl diacylglycerol (α-MGalDAG, 36.1%), phosphatidylcholine (PC, 11.3%), and phosphatidylglycerol (PG, 10.5%) as the major lipids in B. burgdorferi (Hossain, Wellensiek et al. 2001). Also, a cholesteryl galactoside with an immunogenic motif has been described in B.

burgdorferi (Ben-Menachem, Kubler-Kielb et al. 2003; Schroder, Eckert et al.

2008). Previous studies also showed that the membranes of the relapsing fever species B. hermsii contain MGalDAG, PC, PC, cholesteryl glucoside, and an acylated cholesteryl glucoside (Livermore, Bey et al. 1978). Diglyceride-based glycolipids, like the major MGalDAG of the B. burgdorferi envelope, are most widespread in Gram-positive bacteria and mycoplasmas, but essentially absent among Gram-negative bacteria, with the exception of many photosynthetic species (including plant chloroplasts) and some Pseudomonas species (Shaw

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1970). Recently, the monogalactosyl-1,2-diacylglycerol synthase from B.

burgdorferi (bbMGS) was identified, cloned, and functionally verified to show

that this enzyme catalyzes synthesis of the major membrane lipid MGalDAG. Interestingly, the characterized enzyme is closely related to many Gram-positive analogues (Östberg, Berg et al. 2007). Borrelia spirochetes contain an unusual abundance of proteins covalently modified by lipids, i.e., lipoproteins (Haake 2000). A recent re-evaluation using a modified prediction algorithm identified up to 127 lipoproteins in B. burgdorferi genome, corresponding to 7.8% of open reading frames (Setubal, Reis et al. 2006). This proportion is significantly higher than in other complete bacterial genomes such as Treponema pallidum (2.1%) or Helicobacter pylori (1.3%) (Fraser, Casjens et al. 1997; Casjens, Palmer et al. 2000). Surface-exposed lipoproteins also play an important role in adaptive responses and pathogenicity of Borrelia spirochetes. The most abundant and best studied outer membrane proteins of Borrelia spirochetes are lipoproteins called outer surface proteins (Osps). Osps have very little sequence or structural homology to any other known proteins. Characterization of Osps in different B.

burgdorferi sensu lato strains revealed considerable heterogeneity within and

among different species. The most studied Osps are OspA, OspB, OspC, OspE/Erps/CRASPS, and the Dbps. The reciprocal expression of OspA/B and OspC during Borrelia transition from its tick vector to the mammalian host represents how stage-specific protein expression can contribute to pathogenesis during the natural cycle of spirochetal transmission. In unfed ticks,

Borrelia expresses large amounts of OspA and OspB and almost no OspC. The

production of OspA and OspB proteins decreases in the mammalian host and instead the spirochete promotes production of OspC (Schwan, Piesman et al. 1995; Ohnishi, Piesman et al. 2001). Vaccination with a rOspA protects against subsequent B. burgdorferi infection (Wheeler, Garcia Monco et al. 1993). Despite its efficacy, it is not used as a vaccine anymore, in part because of assumed side-effects (Lathrop, Ball et al. 2002). Immunization with OspC also confers protective immunity in animal studies (Steere, Sikand et al. 1998).

General characteristics of porins

In contrast to E. coli, the fluid and fragile outer membrane of Borrelia contains very low amounts of integral membrane proteins (Brandt, Riley et al. 1990; Walker, Borenstein et al. 1991; Radolf, Bourell et al. 1994). The integral outer

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23 | P a g e membrane proteins partly maintain bacterial cell structure, bind to different substrates, are involved in solute or protein transport, signal transduction, interaction with cells and fulfill a number of other tasks important for bacteria (Achouak, Heulin et al. 2001). Secretion or uptake of nutrients through the membranes is accomplished by several pore-forming proteins (Nakae 1975; Benz 1994) that can be divided into three different classes: general diffusion pores, specific channels containing a binding site for a certain solute, and actively transporting, energy-consuming channels. In recent years, numerous structures of outer membrane proteins were established by crystallography, which lead to further subdivision of these proteins into five groups (Schulz 2004). These include general porins, the most abundant species of outer membrane proteins. A good example is E. coli OmpF (Cowan, Schirmer et al. 1992), which allows the unspecific diffusion of hydrophilic molecules and may show a slight selectivity towards ions. The specific porins are structurally closely related to the general porins although their β-barrels have two more strands, as in the case of E. coli LamB (Schirmer, Keller et al. 1995). A third, distinct group consists of composed pores, where the β-barrel itself is a homo-oligomer, such as a dimer in gramicidin A (Ketchem, Hu et al. 1993), a heptamer in hemolysin (Song, Hobaugh et al. 1996) and trimer in TolC (Koronakis, Sharff et al. 2000). The group of outer membrane transport proteins has a much larger β-barrel and additional internal domains. They do not form a pore, but open and close for the passage through the outer membrane of relatively large molecules, such as iron-containing siderophores, in case of FhuA (Locher, Rees et al. 1998) and cobalamins, in the case of BtuB (Chimento, Mohanty et al. 2003). The fifth group of outer membrane proteins (poreless Omps) contains small β-barrels that are not likely to form a pore, rather they function as membrane anchors, such is OmpA in E. coli (Pautsch and Schulz 1998).

The primary sequence of the vast majority of outer membrane porins does not show any indication of α-helical structures, although it represents the typical structure for membrane proteins (Kyte and Doolittle 1982). As a general rule, the outer membrane porins are trimers formed by monomers consisting 16- or 18-stranded β-barrels, whereas the inner membrane proteins consist of mostly α-helices. The cylindrical structure implies that on average every second amino acid in the outer membrane spanning β-sheets is hydrophobic because it faces the lipid membrane or is hydrophilic and points towards the channel interior

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24 | P a g e

(Vogel and Jahnig 1986). Another structural feature of porins is the inward folded loop attached to the inner side of the barrel wall, which constricts the internal diameter and stabilizes the porin β-barrel. Residues located in this loop determine the channel properties, like the diameter, ion selectivity or substrate affinity (Bauer, Struyve et al. 1989; Jordy, Andersen et al. 1996). Interestingly, these loops show very high sequence variation among homologous porins that might aid in evasion of serum antibodies, bacteriophages or proteases. Although porins show very low sequence homology, the similarities in topology and charge distribution are striking (Schirmer 1998). For instance, the C-terminal transmembrane segment contains an aromatic amino acid that is absolutely conserved in classical porins of Enterobacteriaceae (Struyve, Moons et al. 1991). Yet, this is not the case for the spirochetal porins characterized to date (Nikaido 2003).

In addition to transport of solutes, some porins can have other functions. They can insert into eukaryotic cell membranes (Rudel, Schmid et al. 1996), activate complement (Alberti, Marques et al. 1996), act as receptors for bacteriophages (Yu, Ding et al. 1998) or be targets for bactericidal compounds (Sallmann, Baveye-Descamps et al. 1999). Moreover, surface-exposed regions of porins can adhere to cellular receptors, a feature observed for Shigella flexeneri OmpC (Bernardini, Sanna et al. 1993), Treponema denticola Msp (Fenno, Muller et al. 1996) and B. burgdorferi P66 (Coburn and Cugini 2003).

Characterization of porins

There are several techniques used to study function and properties of porins in

vitro. Among others, reconstitution systems such as the liposome-swelling assay

(Nikaido and Rosenberg 1981) and measurements of exclusion limits (Nakae and Nikaido 1975) are used. Conductance recording across a planar lipid bilayer (also called a black lipid bilayer) is the technique used the most for an initial structural and functional study of membrane channels (Benz, Janko et al. 1978; Schindler and Rosenbusch 1978). Reconstitution experiments aim to reestablish the function of purified porins, demonstrating the integrity of the pore-forming complex after the isolation process. The bilayers can be built as planar membranes or vesicles. Black lipid membranes separating two aqueous phases are particularly stable for electrical measurements (Benz and Janko 1976). The

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25 | P a g e apparatus used for membrane formation consists of a Teflon chamber with a thin wall containing a hole with a diameter of 0.2–1 mm separating the two aqueous compartments. For membrane formation, lipid solution is painted over the hole to form a thick, colored lamella. The experiments can be started after the lamella thins out and turns optically black in reflected light, because the lipid bilayer is much thinner than the wavelength of visible light (Dilger and Benz 1985). The lipid bilayer technique allows very sensitive detection of current through the membrane, which gives the opportunity to measure membrane channel conductance with high accuracy. The bilayer itself has a very low conductance below 10 pS. The membrane current is measured with an electrode pair switched in series with the voltage source and a high sensitivity current amplifier. The addition of a channel-forming protein to the aqueous phase results in a stepwise increase of membrane conductance as a consequence of protein reconstituting into the lipid bilayer membrane and can be recorded. The single channel conductance for an individual porin is usually homogenous and correlates with the size of the channel formed (Trias and Benz 1994). Artificial lipid bilayer measurements in addition to rough estimates of the channel size can provide important data on the electrostatic properties of the pore, such as ion selectivity determined upon permeability ratio of cations over anions (Schirmer 1998).

Porins in spirohetes

The first spirochetal protein reported to have pore-forming activity was a 36.5-kDa major protein of the outer membrane from Spirochaeta aurantia. In its native state, it forms trimers and exhibits 7.7-nS channel-forming activity in the planar lipid bilayer assay (Kropinski, Parr et al. 1987). Oligomeric forms have been observed for several spirochetal porins (Egli, Leung et al. 1993; Shang, Exner et al. 1995; Blanco, Champion et al. 1996). The 53-kDa major surface protein (Msp) from Treponema denticola has adhesin properties and porin activity. Interestingly, it was visible as a hexagonal array on the surface of T.

denticola cells by electron microscopy (Egli, Leung et al. 1993; Mathers, Leung

et al. 1996). Porin activity has also been demonstrated for native, rare outer membrane protein 1 (Tromp 1) of T. pallidum, exhibiting single channel conductance of 0.7 nS (Blanco, Champion et al. 1996). Purified recombinant OmpL1 of pathogenic Leptospira was reconstituted into planar lipid bilayers and

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26 | P a g e

demonstrated 1.1 nS average conductance (Shang, Exner et al. 1995). Up-regulation of OmpL1 porin expression after excretion of virulent Leptospira from the host and following culture-attenuation suggests that OmpL1 plays an important role in the adaptive response (Haake, Champion et al. 1993; Shang, Exner et al. 1995). Borrelia, due to its limited metabolic and biosynthetic capacities, is highly dependent on nutrients provided by their hosts (Fraser, Casjens et al. 1997; Casjens, Palmer et al. 2000). Consequently, these parasites need to have efficient regulation of the nutrient uptake across the cell envelope. The first indication of porins in B. burgdorferi came through investigation of channel-forming activities in the planar lipid bilayer assay of outer membrane vesicles. Two porin activities of 0.6 nS and 12.6 nS were found (Skare, Shang et al. 1995). Subsequent work has so far characterized these possible porins in B.

burgdorferi: P13 and its paralog A01 (Östberg, Pinne et al. 2002; Pinne, Östberg

et al. 2004; Pinne, Denker et al. 2006) and P66 (Skare, Mirzabekov et al. 1997). In this thesis additional three Borrelia pore-forming activity exhibiting proteins Oms38, DipA and BesC are described (Paper I, II and III). Another protein, Oms28, exhibits channel-forming activity but has later been shown not to be an outer membrane pore. Both its localization in the membrane and its function has been challenged. This protein, now designated BBA74, was shown to be a periplasmic outer membrane associated protein that lacks typical porin properties (Mulay, Caimano et al. 2007). In contrast to Lyme disease Borrelia species, knowledge of porin activities in relapsing fever Borrelia is rather limited. There are indications of several pore-forming activities in outer membrane preparations of relapsing fever spirochetes (Shang, Skare et al. 1998; Thein, Bunikis et al. 2008) and genes with high homology to B. burgdorferi p13, oms28 and p66 can be found in the published genomes of the relapsing fever agents B.

duttonii, B. recurrentis and B. hermsii. There are also indications of

uncharacterized channel-forming activities present in B. burgdorferi (Östberg, Pinne et al. 2002).

P66

P66 was the second porin identified in the outer membrane of Lyme disease B.

burgdorferi with single-channel conductance measuring 10 nS (Skare,

Mirzabekov et al. 1997). In recent years P66 has been well studied and identified in both Lyme disease and relapsing fever spirochetes (Bunikis, Noppa

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27 | P a g e et al. 1995; Casjens, Palmer et al. 2000; Pinne, Thein et al. 2007; Lescot, Audic et al. 2008). Its atypical huge conductance would represent a large channel that could accommodate large solutes and facilitate their transport into the bacterial cell. Peptides, long chain fatty acids, deoxynucleotides and other large molecular weight solutes have no obvious outer membrane transport systems and are proposed to diffuse across the outer membrane of B. burgdorferi. Because of the large size pore formed by P66, it seems likely that this protein may play an important role in the passive transport of some of these solutes. Additionally, using a phage display library of B. Burgdorferi, Coburn et al. were able to identify P66 as an outer membrane adhesin that binds integrin (Coburn, Chege et al. 1999). Subsequent experiments with a P66 knock-out strain showed that the loss of this porin abolished integrin binding activity (Coburn and Cugini 2003). Therefore, in addition to pore-forming activity, P66 presumably functions as an integrin binding protein. This same dual function was demonstrated for the 64-kDa and 53-kDa (Msp) proteins from T. denticola. Both proteins have porin activity while the 64-kDa protein is also involved in binding of T. denticola cells to gingival fibroblasts in addition to the ability to bind various extracellular matrix proteins (Fenno, Muller et al. 1996; Fenno, Wong et al. 1997). P66 contains surface-exposed domains (Bunikis, Noppa et al. 1995; Bunikis, Noppa et al. 1996) which exhibit immunogenic potential (Barbour, Jasinskas et al. 2008). An additional function of P66 might be interaction with surface-localized lipoproteins. Osp proteins, in particular OspA, have been shown to limit the access of both antibodies and trypsin to the major surface loop of P66. Thus, the possible close contact between P66 and OspA, and other Osp proteins may hinder accessibility to the P66 protein (Bunikis and Barbour 1999). Further characterization of biophysical properties of P66 and its homologues from Lyme disease and relapsing fever borreliae is described in Paper IV. The atypical huge single-channel conductance of this interesting porin raised a question: Is the surprisingly high conductance due to a similarly outstanding huge channel diameter? The attempts to answer this question are described in Paper V. Even though knowledge about transport across the outer membrane of Borrelia is increasing, a great deal still remains to be determined.

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28 | P a g e

Efflux through channel-tunnels

Export systems

In addition to the inner membrane, exported substances like proteins or drugs have to pass the periplasmic space and have to overcome the second permeability barrier, the outer membrane. In the case of protein export out of the cell, one can distinguish roughly between dependent and sec-independent export pathways. In the sec-dependent export pathways, proteins with a cleavable N-terminal secretion signal are directed to the sec system in the inner membrane, which transports them into the periplasmic space (Fekkes and Driessen 1999; Manting and Driessen 2000). This route is also known as the general secretion pathway (GSP). A database search showed that homologues of all essential components of the sec translocase complex are present in Borrelia (Fraser, Casjens et al. 1997). After or during translocation, the secretion signal is cleaved, resulting in processed periplasmic intermediates. For further export across the outer membrane there are six different mechanisms: the chaperone/usher pathway, the single accessory pathway, the autotransporter, the type II secretion system, the type IV pilus biogenesis system and the type IV secretion system (Henderson, Navarro-Garcia et al. 1998; Konninger, Hobbie et al. 1999; Nunn 1999; Cao and Saier 2001; Sandkvist 2001; Thanassi, Stathopoulos et al. 2002).

There are two distinct sec-independent secretion machineries. Both systems form assemblies which span the whole cell envelope and export proteins without a periplasmic intermediate. The type III secretion system forms the injection needle of pathogens and the flagellar export apparatus. These are complex assemblies formed by up to 20 proteins (Hueck 1998; Young, Schmiel et al. 1999). The other sec-independent export apparatus is the type I secretion system.

The export of drugs and cations is mediated by several distinct transporters in the inner membrane. They can be divided into five families (Paulsen, Chen et al. 2001; Saier and Paulsen 2001). One group belongs to the ancient superfamily of ATP-binding cassette (ABC) transporters using ATP as an energy source. The other four families are energized by proton motive force and act as proton antiporters. They belong to the major facilitator superfamily (MFS), the small

Borrelia channel-forming proteins : structure and function